AU721530B2 - Endothelial lining effects and treatment of vasospastic disorders - Google Patents

Abstract

Vasospastic disorders such as Raynaud's phenomenon are treated by administering to the patient an aliquot of autologous blood which has been subjected simultaneously to heat, ultraviolet irradiation and oxidative environment so as to render the blood aliquot capable of generating in the patient's bloodstream after administration a population of stimulated leukocytes having upregulated expression of CD25 cell surface markers, along with an enhancement of endothelial function in the patient.

Description

WO 96/34613 PCT/CA96/00282 ENDOTHELIAL LINING EFFECTS AND TREATMENT OF VASOSPASTIC DISORDERS FIELD OF THE INVENTION This invention relates to treatment of blood cells, and the use of treated, modified blood cells in connection with certain abnormal mammalian physical conditions and disease states. More specifically, it relates to modified mammalian blood and blood cells, methods of achieving the modified mammalian blood and blood cells, and the treatment, among other disorders, of vascular disorders associated with deficient endothelial function, such as vasospatic disorders, in a mammalian patient by administration to the patient of such modified blood and blood cells.

BACKGROUND OF THE INVENTION The control and regulation of blood flow through the cardiovascular system of a mammal is well recognized to be of importance in connection with cardiovascular disorders -such as atherosclerosis, peripheral vascular disease and many other circulatory disorders. There is an emerging body of literature which indicates that the endothelium plays a major role in the regulation of blood flow through the cardiovascular system. The endothelium is a cellular structure which lines the blood vessels, communicating with the smooth muscle layer of the blood vessel walls. Contraction of this muscle layer causes the blood vessels to constrict (vasoconstriction), and relaxation of this muscle layer causes the blood vessels to expand (vasodilation). A normally functioning endothelium effectively controls the smooth muscles of the vessel wall, by secreting vasodilators or vasoconstrictors which diffuse or are carried to the muscle fibres to cause the muscle fibres either to relax or to contract. One such vasodilator secreted by the endothelium is commonly referred to as "endothelium derived relaxing factor" (EDRF), but has recently been established to be nitric SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCTCA96/00282 2oxide, a form thereof or a closely related compound. In addition to regulating blood flow,nitric oxide is recognized as having many other actions within the body, including neurotransmis-sion, a smooth muscle controlling function within the gastro-intestinal tract, both natural and drug induced analgesia, a role in impotence and in tumour toxicity.

It is believed that defective functioning of the endothelium of a patient is an underlying factor in many cardiovascular diseases observed in mammalian patients.

For example, a patient with atherosclerosis has excessive quantities of lipid underlying the endothelium, including oxidized low density lipoprotein (containing cholesterol) which is believed to interfere with the proper functioning of the endothelial cells.

SUMMARY OF THE INVENTION The present invention is based upon the discovery that one can extracorporeally subject an aliquot of a mammalian patient's blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, to certain stressors and create in the aliquot modified leukocytes. On re-introduction of the aliquot to the patient's body by various routes including intra-muscular injection, the modified leukocytes have certain beneficial effects. One of these effects is stimulation of the activity of a functionally deficient endothelium.

The modified leukocytes of the present invention can be obtained by subjecting the aliquot of the patient's blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, to stressors selected from heat, ultraviolet radiation and oxidative environments such as treatment with ozone/oxygen SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 -3 mixtures, or any combination of such stressors, simultaneously or sequentially.

Thus, according to the present invention, ex vivo treatment of the blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, promotes the subsequent development of modified leukocytes, which are different from the untreated cells that are found in the peripheral blood of normal individuals and patients before receiving this treatment, in that they cause an in vivo effect.

The effects of the modified leukocytes of the present invention, when re-injected into the mammalian patient's body, are several in number. Firstly, there is an effect on other leukocytes or their progenitors which have not been modified by the externally applied stressors, as a result of cell-to-cell communication, a widely recognised phenomenon among cells of the immune system. The result of injecting the blood subjected to stressors outside the body is the upregulation of specific cell surface markers such as HLA-DR and CD25 on other, nontreated leukocytes in the peripheral blood, circulating in the patient. This is indicative of an enhanced immune system. It appears that the treated leukocytes release cytokines (intercellular messenger peptides and proteins), or stimulate leukocytes of the recipient to do so, initiating a cascade phenomenon which affects a number of the quiescent leukocytes in the peripheral blood and causes them to become stimulated. This apparently leads to improved blood flow at sites in the body far removed from the site of injection of the treated leukocytes.

Secondly, the stimulated leukocytes present in the blood circulation, perhaps through the intermediary of the same or similar cytokines and probably after physical contact or binding to the endothelium via cell adhesion SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCTI/CA96/00282 4 molecules, act upon the endothelium, either directly or indirectly, to increase the endothelial vasodilator function probably by increasing the production and/or action of vasodilators such as nitric oxide, prostacyclin and/or by inhibiting the production and/or action of vasoconstrictors, so as to increase blood flow. This can be manifested either as a restoration of the function of a portion of the endothelium which has become defective, the portion being close to or remote from the site of injection. Such restoration of function may occur through repair of deficient cells or an enhanced rate of replacement of damaged cells. It can also be manifested as an overall improvement in endothelial function. This increased blood flow resulting from increased endothelial vasodilator function, and the consequent increase in oxygenation of tissues, is indicative of use of the process of the present invention in treatment of patients with vascular disease including those with advanced peripheral vascular disease, those with chronic varicose ulcers, and those at risk of developing gangrene which frequently results in amputation. In general, the leukocytes according to the present invention are indicated for use in treating any type of vascular disease either involving partial or complete blood vessel occlusion leading to restricted blood flow or dysfunction of the mechanisms required to permit adequate vasodilation such that tissues including those of the central nervous system, heart, lungs, gastrointestinal tract, liver, kidneys, placenta or extremities would be acutely or chronically affected in terms of structure or function.

BRIEF REFERENCE TO THE DRAWINGS FIGURE 1 is a diagrammatic illustration of the believed mode of action of the injected leukocytes according to the present invention, in the vascular system.

SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 5 FIGURES 2 and 3 are graphical presentations of the results of clinical tests of the use of the present as described in Example 3 below.

DESCRIPTION OF THE PREFERRED EMODIMENTS With reference to the Figure i, there is diagrammatically illustrated an arteriole 10. An arteriole is a blood vessel forming part of the mammalian vascular system, of a size smaller than an artery, and which receives circulating blood from an artery, fed from the heart. Arterioles gradually decrease in size, in the downstream direction. A metarteriole 12, which is a smaller terminal branch of the arteriole 10, is shown branching off the arteriole 10. Metarterioles such as 12 lead to capillaries which are the site of nutrient and gaseous exchange between the blood and the tissues, with oxygen diffusing from the blood to the tissues and carbon dioxide diffusing from the tissues to the blood. The arteriole 10 is lined with a cellular layer of endothelium 14. Surrounding the exterior of the arteriolar endothelium 14 is a layer of smooth muscle 18, which can contract or expand to cause alterations in the size of the arteriole causing either vasodilation to increase the amount of blood flow therethrough or vasoconstriction to reduce the amount of blood flow therethrough. The metarteriole 12 is similarly lined with endothelium 16 and has a single layer of smooth muscle 19, functioning similarly.

As in the case of all the other blood vessels in the body, of all the various sizes, capillaries are lined with endothelium. However, there is no smooth muscle layer associated with the capillaries.

According to the preferred process of the present invention, an aliquot of blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 6 including platelets, is extracted from the patient and treated with certain stressors, described in more detail below. The effect of the stressors is to modify leukocytes in the aliquot. These modified leukocytes 20, along with the rest of the aliquot, are re-injected intramuscularly and, after a period within the muscle, of uncertain and probably variable duration, they probably gain entry to the general circulation by passing through the blood vessel wall, where mixing with the blood occurs. Downstream from the point of entry 22, therefore, the blood will contain modified leukocytes 20 in addition to unmodified leukocytes 24, red blood cells 26 and platelets 28, as well as other components.

The injection of modified leukocytes 20 appears to have a number of unexpected effects on the arteriole and on the blood flowing through it. One effect is the stimulation of quiescent leukocytes 24, which have not been subjected to the stressors outside the body, to transform into stimulated leukocytes 30. This activation process may occur in sites other than the blood vessels, for example in immune system organs such as lymph nodes, spleen or bone marrow, and may involve leukocyte progenitors in addition to or alternatively to mature leukocytes. Thus a cascade mechanism is set in motion.- This is believed to be effected by the secretion of certain cytokines 32, the precise nature of which is uncertain, either from the modified leukocytes 20 themselves or by the stimulation of endogenous leukocytes 30 by direct cell-cell interaction.

These mechanisms serve to effect stimulation of previously quiescent leukocytes or progenitors to create the stimulated leukocytes 30. The stimulated leukocytes probably adhere to the damaged endothelial cells via cell adhesion molecules such as ICAM-1 expressed on the endothelial cells interacting with activated LFA-l expressed on certain activated leukocytes. Other cytokines 34, perhaps originating from these adherent leukocytes and SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCIT/CA96/00282 7 which may in fact be the same as or different from the cytokines 32, contact the endothelium 14, and cause it to secrete vasodilators 36 which have the effect of relaxing the smooth muscle 18 to cause a degree of vasodilation.

Other secretions 38 released from the endothelium 14 by the cytokines 34, which may or may not be the same substances or mixture of substances as the vasodilators 36, contact the platelets 28, and have the effect of inhibiting their ability to aggregate. Whilst the accompanying figure diagrammatically illustrates endothelial effects at the endothelium of an arteriole of the vascular system, it appears that the process of the invention has a similar effect on endothelial cells at all levels of the vascular tree.

It is believed that one of the components of the vasodilators 36, and one of the components of the secretions 38, is endothelium derived relaxing factor EDRF, probably nitric oxide or closely related substances, and a second component is prostacyclin. Alternative or additional sources of nitric oxide and/or other vasodilators resulting from a treatment according to the present invention may be stimulated leukocytes present in the circulation or stimulated platelets present in the circulation.

The stressors to which the leukocytes are subjected ex vivo according to the invention are selected from temperature stress (blood temperatures above or below body temperature), an oxidative environment and ultraviolet radiation, individually or in any combination, simultaneously or successively. The leukocytes are subjected to the stressors as a part of an aliquot of mammalian blood or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, extracted from the patient to whom the aliquot is to be injected following subjection to the stressors.

SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 8 Suitably the aliquot has a volume of from about 0.1 100 mis, preferably 5 15 mis, a most preferred, typical aliquot being 10 mis in volume.

It is preferred, according to the invention, to apply all three of the aforementioned stressors simultaneously to the aliquot under treatment, in order to ensure the appropriate modification of a sufficient number of leukocytes. Care must be taken not to utilised an excessive level of the stressors, to the extent that the cells or any parts of them become irreparably damaged, or that the cell membrane is caused to be disrupted.

The temperature stressor, when it is applied, must keep the aliquot liquid, and should not heat it above about 55 0 C, for fear of damaging the cells. Preferably the temperature stressor warms the aliquot being treated to a temperature above normal body temperature, to about 37 55 0 C, and most preferably from about 40 50 0

C.

The oxidative environment stressor can be the application to the aliquot of liquid or gaseous oxidizing agents. Preferably it involves exposing the aliquot to a mixture of medical grade oxygen and ozone gas, most preferably by bubbling through the aliquot, at the aforementioned temperature range, a stream of medical grade oxygen gas having ozone as a minor component therein.

Suitably the gas stream has an ozone content of from about 100 Ag/ml, preferably 3 70 gg/ml, and most preferably about 5 50 pig/ml. The gas stream is supplied to the aliquot at a rate of from about 0.01 litres/minute, preferably at about 0.05 5.0 litres per minute and most preferably at about 0.06 litres per minute

(STP).

The ultraviolet radiation stressor is suitably applied by irradiating the aliquot under treatment from an SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCTCA96/00282 9appropriate source of UV radiation while the aliquot is maintained at the aforementioned temperature and while the oxygen/ozone gaseous mixture is being bubbled through the aliquot. Appropriate UV sources are UV lamps emitting in the C-band wavelengths, i.e. at wavelengths shorter than about 280 nm. Ultraviolet radiation corresponding to standard UV-A and UV-B sources can also be used. Emissions at wavelength 253.7 nm are particularly suitable. An appropriate dosage of such UV radiation, applied simultaneously with the aforementioned temperature and oxidative environment stressors, is obtained from lamps with a power output of from about 15 to about 25 watts, at the chosen UV wavelength, arranged to surround the sample container holding the aliquot, each lamp providing an intensity, at a distance of 1 meter, of from about 45 mW/sq.cm. Up to eight such lamps surrounding the sample bottle, with a combined output at 253.7 nm of 15 watts, operated at an intensity to deliver a total UV light energy at the surface of the blood of about 0.025 preferably about 0.1 0.5 joules/cm 2 may advantageously be used. Such a treatment provides an aliquot which contains stimulated leukocytes according to the invention in appropriate amounts to cause the effects outlined above ready for re-injection into the patient.

The time for which the aliquot is subjected to the stressors is normally within the time range of from about 0.5 60 minutes. This depends to some extent upon the chosen intensity of the UV irradiation, the temperature and the concentration of and rate at which the oxidizing agent is supplied to the aliquot. The more severe the stressors applied to the aliquot, generally the shorter time for which they need to be applied. Some experimentation to establish optimum times may be necessary on the part of the operator, once the other stressor levels have been set. Under most stressor conditions, preferred times will be in the approximate range 2 5 minutes, and SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 10 normally around 3 minutes. The starting blood temperature, and the rate at which it can be warmed or cooled to a predetermined temperature, tends to vary from patient to patient.

In the practice of the preferred process of the present invention, the blood aliquot (or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, these various leukocyte-containing combinations, along with whole blood, being referred to collectively throughout as the "aliquot") may be treated with the stressors using an apparatus of the type described in U.S. patent 4,968,483 Mueller. The aliquot is placed in a suitable, sterile, radiationtransmissive container, which is then fitted into the machine. The temperature of the aliquot is adjusted to the predetermined value, e.g. 42.5 0 C, and the UV lamps are switched on for a fixed period before the gas flow is applied to the aliquot providing the oxidative stress, to allow the output of the UV lamps to stabilize. Then the oxygen/ozone gas mixture, of known composition and controlled flow rate, is applied to the aliquot, for the predetermined duration of 0.5 60 minutes, preferably 2 minutes and most preferably about 3 minutes as discussed above, so that the aliquot experiences all three stressors simultaneously. In this way, appropriately modifieded leukocytes according to the present invention are generated in numbers sufficient to achieve the desired effects.

Another characteristic of the modified leukocytes of the present invention is their ability to generate in the bloodstream of the patient, after re-injection, stimulated leukocyes having an increase in activation markers, for example CD 25 (IL-2 receptor), Ber-Mac 3 and HLA-DR (T-cell and monocyte activation). These cell surface markers are surface proteins, the quantities of which are increased by injecting the modified leukocytes prepared SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCTICA96/00O8Z 11 using the stressors of the preferred process of the present invention i.e. temperature, an oxidising environment, and UV radiation. They can be quantified by staining techniques and use of conventional monoclonal antibody techniques.

The proper functioning, or lack thereof, of the endothelium of a patient, at a particular location, can be tested by using a method which involves the iontophoretic introduction of acetylcholine through the skin, and measurement of its effect on superficial blood at the chosen location.

Acetylcholine added to a blood vessel which has intact, properly functioning endothelium, stimulates the production and secretion of nitric oxide by the endothelium. The nitric oxide so produced acts as a vasodilator and causes the smooth muscle cells to relax.

The effect of such vasodilation can be quantitated by measuring the flow of blood in the vessel, e.g. by laser Doppler flowmetry. If, however, the endothelium of the patient has a major functional defect, the acetylcholine may act directly on the smooth muscles themselves, and cause them to contract, resulting in vasoconstriction, the opposite of the effect observed when the patient's endothelium is functioning normally.

Endothelial function may accordingly be examined clinically as follows. Acetylcholine is applied to the skin of the patient and a small electric current is applied across the skin between two adjacent electrodes, one positively and one negatively charged (iontophoresis).

Acetylcholine, being a charged molecule, passes through the skin with the current to the superficial blood vessels immediately below the skin surface. At this site, acetylcholine acts on the endothelial cells of the blood vessels, stimulating them to produce EDRF nitric SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/0082 12 oxide), resulting in vasodilation (endothelial dependent vasodilation) and an increase in blood flow, which is measured by laser Doppler flowmetry, for example. This technique is known, in general terms see, for example, Chowienczyk et.al., "Impaired endothelium-dependent vasodilation of forearm resistance vessels in hypercholesterolaemia", The Lancet, Vol. 340, December 12, 1992, p.1430.

A specific preferred application for the present invention is in treatment for, or at least alleviation of the symptoms of, the vasospastic type diseases as exemplified by Raynaud's Phenomenon and other vasospastic disorders such as migraine, cluster headache and syndrome X. Raynaud's Phenomenon is a state in which the fingers and/or toes undergo a series of colour changes, white (vasospasm), then blue (capillary stagnation) then red (arterial dilation), lasting a variable time and for a variable frequency, and with or without pain. Raynaud's Disease or primary Raynaud's is the term for Raynaud's Phenomenon in the absence of any other detectable disease.

Raynaud's syndrome, or secondary Raynaud's, is the term for Raynaud's Phenomenon associated with other diseases.

Whilst Raynaud's is not in itself life threatening, it may lead to the onset of other pathological conditions, and to-date there has been no effective cure put into general practice for it. A patient suffering from Raynaud's must simply endure the associated pain and discomfort. In a small proportion of cases, the ischemia of the digits resulting from extreme vasospasm may lead to skin ulceration and gangrene, which may result in amputation. Raynaud's Phenomenon can affect patients at substantially any age, and affects about four times as many women as men.

SUBSTITUTE SHEET (RULE 26) 13 Following one or more treatments according to the procedure of the invention, spaced at appropriate intervals, dramatic alleviation of the symptoms of the Raynaud's phenomenon is experienced, in the cases of all of the patients on which the treatment has so far been employed. Iontophoretic measurements conducted on the blood flow of the subject patients, before and after undergoing the treatment according to the invention, have shown an enhancement of the endothelial performance in the patients of concern, and an alleviation of the Raynaud's phenomenon symptoms.

Thus from another aspect, the present invention provides an aliquot of autologous human blood for administration to a patient to alleviate Raynaud's phenomenon in the patient, the aliquot of autologous blood having been subjected, in vitro, to at least one stressor selected from heat, UV radiation and ozone/oxygen mixture to stimulate leucocytes therein, to the extent that, upon re-injection into the patient, a significant enhancement of 20 endothelial function to cause vasodilation is experienced.

Throughout the description and claims of this specification, the word "comprise" and variations of the word such as "comprising" and "comprises", means "including but not limited to and is not intended to exclude other additives, components, integers or steps".

o The invention is further illustrated and described with reference to the following specific examples.

EXAMPLE 1 A patient suffering from primary Raynaud's disease was given a course of treatment under carefully controlled clinical conditions, according to the present invention. Each treatment administered to the patient involved removing a small aliquot of the patient's blood, exposing it to stressors ex vivo and re-introducing the treated blood aliquot to the patient as described in the invention. The course of treatment administered to the patient comprised ten such individual treatments, one H:\WendyS\Keep\species\54944-96 Vasogen.doc 19/04/00 WO 96/34613 PCT/CA961002S2 14 administered each day over a period of about two weeks.

Raynaud's disease is a disorder associated with peripheral vasospasm and inadequate blood flow at the extremities of the patient's body, such as the fingers, characterized initially by marked pallor of the fingers on exposure to cold, often followed by a subsequent phase of cyanosis (bluish colour) and a slowing of the restoration of blood flow after cold exposure ceases.

The subject of this experiment was a female patient of about 24 years of age who had suffered from primary Raynaud's disease for at least 10 years, with the fingers of both hands being severely affected. Prior to commencing the course of treatments according to the invention, the blood flow in the superficial blood vessels of the fingers was measured by laser Doppler flowmetry and photoplethysmography in response to mild cold and warm stresses of the hand. The skin surface temperatures of the fingers were also measured by a thermographic technique in response to warm and cold stress of the hands. The blood flow in response to iontophoretic introduction of acetyl choline was measured by laser Doppler flowmetry, prior to commencing the course of treatments.

mls of the patient's blood were extracted from an arm vein, anticoagulated and collected in a sterile sample bottle. This sample was subjected to heat UV radiation oxidising (oxygen/ozone mixture) stressors according to the procedure of the invention, in an apparatus generally as illustrated in aforementioned U.S.

Patent 4,968,483 Mueller. The blood sample was heated to 42.5 0 C and UV irradiation of wavelength 253.7 was applied to it. Upon reaching the required temperature (42.5 0

C),

a gaseous mixture of medical grade oxygen with an ozone content of 12.5 micrograms per ml, at a gas flow rate of about 60 ml/min was bubbled through the sample for 3 min.

SUBSTITUTE SHEET (RULE 26) WO 96/34613 PC/CA96/00282 15 After the ex vivo treatment of the blood sample had been completed according to the procedure of the invention, the sample was re-injected into the gluteal muscle of the patient. Shortly after completion of the course of ten treatments over a period of two weeks, the patient reported an alleviation of her symptoms of Raynaud's disease. This state of improvement persisted for at least two months after the completion of the course of treatment.

The skin temperature and the blood flow measurements in response to warm and cold stress and the blood flow after iontophoretic introduction of acetyl choline were repeated after the course of treatment according to the invention. The responses to warm and cold stress were reflective of a more normal response after treatment than before. The blood flow response to acetyl choline after treatment was increased, indicating an increase in the ability of the endothelium to produce vasodilators following the treatment. All the measurements showed evidence of an increase in peripheral blood flow following the treatment along with the alleviation of the symptoms of Raynaud's disease.

Generation in the blood of treated patients of increased levels of prostacyclin is also an effect resulting from the ex vivo stressing of the blood cells in the manner described, according to the present invention.

Amelioration of the effects of a defective endothelium with its consequent alleviation of abnormal conditions associated therewith, such as vasospastic disorders as exemplified by Raynaud's disease, is just one of the several beneficial effects likely to be derived by correct medical use of the processes, techniques and modified leukocytes forming aspects of the present invention.

SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 16 EXAMPLE 2 Four patients, including the subject of Example 1, human females ranging in age from 15 to 84 years, and all suffering from primary Raynaud's phenomenon, were subjected to a course of treatment according to the present invention. Treatment was given by skilled, qualified personnel, in a medical hospital facility on an out-patient basis.

Each treatment administered to a patient involved removing a 10 ml aliquot of the patient's blood, into an apparatus as generally illustrated in aforementioned U.S.

Patent No. 4,968,483 Mueller, heating the sample to 42.5 0

C

and exposing it to UV irradiation at wavelength 253.7 nm.

Upon reaching the required temperature (42.5 0 a gaseous mixture of medical grade oxygen with an ozone content of 12.5 micrograms per ml, at a gas flow rate of about ml/min was bubbled through the sample for 3 minutes.

After the ex vivo treatment of the blood sample had been completed in this manner, the sample was reinjected into the respective patient via the gluteal muscle. Each patient underwent a course of 10 such treatments over a period of 2 4 weeks, the individual treatments being spaced apart by about 1-3 days.

Subjectively, every patient reported a very significant alleviation of her Raynaud's symptoms, after completion of the course of treatments. This state of improvement persisted for at least two months after the completion of the course of treatment. In the case of the 14 year old female patient, she exhibited as part of the Raynaud's symptoms initially, small, slow healing ulcerations of the feet, around the toes. All of these had healed by the end of the course of 10 treatments. The 84 year old patient initially had noticeably disfigured SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 17 fingernails at the fingernail beds, resulting from reduced nutrition and reduced oxygenation caused by lack of circulation of blood thereto. At the end of the course of treatment, new growth of nails from the nail bed had started and was clearly visible.

EXAMPLE 3 In the case of each patient, objective measurements of blood flow, before and after the course of treatments, were made by the iontophoretic technique using acetylcholine previously described. The iontophoretic applications and measurements were made on the patients' forearms. The initial measurements on each patient were taken immediately before the first treatment during visit The subsequent measurements were all taken one day after the completion of the course of ten treatments during visit 11) and again on a follow-up basis two or three weeks later e. during visit 12). One of the four patients was given a second, subsequent course of five further treatments. Blood flow was measured by laser Doppler flowmetry, to give an indication of vasodilation.

For each measurement, a reservoir containing acetylcholine was mounted on the patient's arm with the acetylcholine in contact with the patient's skin.

Electrodes were inserted into the reservoir, so that a current of known but variable magnitude could be applied to the reservoir to exert an iontophoretic effect. The dose of acetylcholine applied to the skin is a function of the time of acetylcholine-skin contact and the voltage applied between the electrodes, thereby giving a dose in arbitrary units.

Figure 2 of the accompanying drawings presents a graph of observed laser Doppler flow of blood, against dosage in arbitrary units determined as above, for one SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCTICA96/00282 18 representative patient. The duration of the iontophoresis was arbitrarily divided into various equal time intervals or epochs. The mean flow at each epoch is plotted against time, with the mean plotted at the mid time point of each epoch. The curve denoted by circles is that obtained from testing conducted before the first treatment, i.e. on the patient's first visit. As shown, blood flow increases in a generally sigmoidal fashion as the acetyl choline dosage (function of contact time and applied iontophoretic voltage) increases. The curve denoted by triangles is that obtained in a similar manner, on the patient's 11th visit, one day after the conclusion of the course of ten treatments. The curve denoted by squares is that obtained on the patient's 12th visit, twenty eight days after the 11th visit. Effectively, these are dose response curves.

A significant increase in blood flow in response to acetylcholine, indicative of an enhanced endothelial function, after the course of treatment, is evident from these curves.

The shapes of the respective curves indicate that increasing doses of acetylcholine cause increasing levels of blood flow, up to a plateau level in each case. This is explained by the fact that the acetylcholine is stimulating the production and secretion of the vasodilator nitric oxide (EDRF) by the endothelium, and that the endothelium is acting normally.

All four of the patients treated showed essentially similar results, those presented on Figure 2 being representative, and from a single patient. Figure 3 of the accompanying drawings shows similar curves to Fig.

2, but derived from the means of the measured blood flows of all four of the patients. As in the case of the Fig. 2 curves from the one patient, the curves denoted by circles are the mean blood flow values, at various, increasing doses of acetylcholine from the four patients before the SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 19 first treatment. The curves denoted by triangles are mean values from one day after the conclusion of the course of treatments. The curves denoted by squares are mean values from twenty eight days after the conclusion of the course of treatments. The dosage response trend is clearly apparent from the curves presented as Fig. 3.

EXAMPLE 4 The iontophoresis data obtained from all four patients as described above, was subjected to statistical analysis, using the data of each of the four patients obtained before any treatment, and the data obtained from all four patients two to three to four weeks after completion of the course of 10 treatments (visit 12).

As noted above, in obtaining the curves shown on Figure 2, the mean flow at each epoch is plotted against time, with the mean plotted at the mid time point of each epoch. Since the graphs indicate that the flow increased in a sigmoid fashion, the slope of the increase was calculated, in each case, using the mean flows from the epoch with a curve starting to rise, to the point where the curve started to become asymptotic. The regression analyses used to calculate these slopes all accounted for greater than 85% of the variation, and were therefore considered a very good fit. There was also calculated a total area under the curve (AUC) from the point where the curve started to rise, to epoch 10. The maximum recorded mean flow and the area under the curve during epoch 11 were also analyzed.

Table 1 summarizes these results. It indicates that the increase in flow in response to acetylcholine was higher post treatment, since the maximum flow, the AUC during the increase and the AUC in epoch 11 were higher post treatment, to a statistically significant extent, even SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 20 on the basis of four patients (the P value being 0.012, 0.020 and 0.040 respectively). The slope was also greater, but not significantly so.

SUBSTITUTE SHEET (RULE 26) TABLE 1.

SUMMARY OF ANALYSIS OF IONTOPHORESIS DATA

C,,

C-

co

C',

m

C-

m

'.D

Baseline Before Visit 12 After Visit 12 Minus 95% P Value from Treatment Course of Baseline Confidence T-Test Treatment Interval Drug Variable Mean SD Mean SD Acetyl Choline, Slope of Increase 36.55 17.360 48.1 12.065 12.26 -1.56,26.08 0.067 Acetyl Choline Maximumn Flow 211.4 40.67 272.3 22.68 60.93 25.96,95.89 0.012 Acetyl Choline AUC during increase 80695 24238.1 106708 15247.0 26013 7899,44127 0.020 Acetyl CholineTAUC in epoch 11 641M3 9 13167.0 83765 6623 1662106,37145 0.040 WO 96/34613 PCT/CA96/00282 22 EXAMPLE Nineteen normal, healthy volunteers, 15 males, 4 females, aged between 20 and 30 years, were enrolled in the study. They were blindly assigned to two groups, 14 males, 4 females) being given the treatment, the remaining males constituting the control group. The volunteers were required to refrain from smoking during the study and to avoid taking drugs likely to affect platelet function, as recommended by the British Society of Haematology (1988).

Individuals were bled by venepuncture, and re-injected with of their own blood on five occasions over a period of two weeks. Thus the injections were administered at 2-3 day intervals according to the following protocol: DAY Bleed vol (ml) Re-iniect vol (ml) Mon 40 Wed 20 Fri 20 Mon 20 Wed 10 Wed(+2h) Fri Individuals in the treatment group received five intramuscular (gluteal) injections of 10mL autologous blood which had been treated as described in Example 1 above.

Individuals assigned to the control group were re-injected with 10mL of their own untreated blood and the study was performed blind, in that neither the subjects, the supervising clinician nor the laboratory staff performing the analyses were aware of the group to which an individual was assigned.

Prostacyclin levels in peripheral blood were estimated from the plasma concentration of its stable metabolite, 6-keto-prostaglandin-Fla (Wu et.al., "Journal SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 23 of Clinical Investigation" 75: 168-174, 1985), using a commercially available radioimmunoassay from Amersham International, U.K. The leukocyte surface markers CD25 (the a-subunit of the IL-2 receptor), the MHC Class 2 antigen HLA-DR and the monocyte-specific Ber-Mac 3 were assessed in mononuclear cells(monoctyes, T-cells and B-cells), isolated by density gradient centrifugation, using immunocytochemical staining with commercially available monoclonal antibodies. Quantitation of CD25, HLA-DR and Ber-Mac 3 positive cells was performed microscopically, with manual counting, and numbers were expressed as a percentage of the leukocyte common antigen positive cells.

Statistical Analysis: Significance was determined using Student's t-test for paired data to compare the posttreatment times with pre-treatment values. Results from control and treated groups of individuals were also compared.

Prostacyclin measured as 6-keto-prostaglandin Fla The results from the treated group and control groups are shown in Tables 2 and 3. Table 4 shows a summary of the statistical analysis of these data. One subject (number 3 was excluded from the date due to cigarette smoking.

Within the treated group, the post-treatment 6keto-PGFla concentrations were significantly (p 0.05) higher on two of the five post-treatment measurement occasions, post-i and post-4, when compared with the pretreatment baseline values. No such significant differences were observed in the placebo-treated control group.

SUBSTITUTE SHEET (RULE 26) WO 96/34613 WO 9634613PCICA96OO282 -24 TABLE 2 The concentrations of 6 -keto-pros taglandin Fla [pg/mL] in peripheral blood from treated volunteers before and after treatment.

Treated Pre- Post-I Post-2 Post-3 Post-4 Subject No. Treatment 1 55.3 68.4 58.2 59.8 101.8 51.4 2 16.3 12.9 14.2 17.2 24.0 25.0 4 16.8 34.0 17.3 51.0 49.0 60.0 17.2 44.4 30.8 25.8 20.0 37.0 6 53.8 56.6 72.0 44.7 88.5 68.8 723.4 34.7 31.8 15.3 24.5 54.6 830.7 27.7 23.6 38.4 34.6 26.4 9 44.1 33.0 38.6 29.7 12.3 21.9 16.1 25.5 20.0 29.4 22.1 15.1 11 10.3 1 17.4 13.1 37.5 11.2 12 43.0 82.8 42.7 107.0 68.2 56.2 13 22.2 24. 1 33.0 15.9 10.6 23.5 14 14.8 17.7 18.4 27.2 47.6 36.7 Mean 28.00 36.54 32.15 36.50 41.59 37.52 TABLE 3 The concentration of [pg/mrL] in peripheral blood from volunteers before and after a untreated blood.

6-keto-prostaglandin Fla placebo- treated (control) course of injections of SUBSTItUTE SHEET (RULE 26) WO 96/34613 PCTCA96/00282 25 Control Pre- Post-1 Post-2 Post-3 Post-4 Subject No. Treatment (Baseline) 1 34.0 29.0 45.0 31.0 12.0 11.0 2 32.0 36.0 12.0 35.5 37.5 30.5 3 52.5 62.5 25.3 18.0 49.0 37.0 4 29.0 28.5 57.0 38.0 44.0 29.5 25.5 96.0 25.2 39.0 33.0 23.0 Mean 34.60 50.40 32.90 32.30 35.10 26.20 TABLE 4 Statistical analysis of the data of Tables 2 and 3.

Treatment Time Baseline 1 2 3 4 Control Baseline 0.3191 0.8742 0.8003 0.941 0.1426 vs Control Significance ns ns ns ns ns Treated Basline 0.0434 0.0648 0.1672 0.0444 0.0687 vs Treated Significance <0.05 ns ns <0.05 NS Treated vs Control 0.3253 0.3758 0.9385 0.6107 0.5359 0.1208 Significance ns ns ns ns ns ns Exbressinn nf rn3f ~n ~~ri~h~rl~ h~ hh~ nl~~ E The proportion of cells expressing CD25, measured as a percentage of all CD45+ mononuclear cells, is shown in Tables 5 and 6 below. Table 7 shows the statistical analysis of these data. The results show that, on each of the five occasions investigated during and after a course of treatment, a significantly higher proportion of peripheral blood mononuclear cells expressed CD25 compared SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 26 to the pre-treatment values. There was no such increased level of expression of this marker in the placebo treated control group. In addition, at post-treatment times 2 and 3 the treated group showed a significantly higher expression of this marker than the control group.

TABLE The proportion of cells expressing CD25, as a percentage of CD45+ cells, in peripheral blood from treated volunteers before and after a course of treatment.

Treated PreTreatment Post-1 Post-2 Post-3 Post-4 Subject No. (Baseline) 1 31 43 51 45 45 2 12 75 39 21 57 74 3 29 69 72 27 100 4 12 22 32 99 12 50 96 89 87 85 71 6 40 42 46 58 43 7 51 50 50 64 47 58 8 47 51 41 46 36 38 9 48 45 46 76 45 52 50 53 77 63 56 82 11 17 26 34 55 58 12 34 32 58 40 31 13 40 42 60 72 56 88 14 47 48 48 53 57 71 Mean 3629 49.57 53.07 57.57 52.00 54.21 SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCTICA96/00282 27 TABLE 6 The proportion of cells expression CD25 (as a percentage of CD45+ cells) in peripheral blood from placebo-treated (control) volunteers before and after a course of injections of untreated blood.

Control Pre- Post-1 Post-2 Post-3 Post-4 Subject No. Treatment (Baseline) 1 47 42 25 32 46 2 48 32 52 28 18 32 3 25 39 35 30 21 34 4 22 73 31 17 45 57 60 49 46 44 40 34 Mean 40.40 47.00 37.80 30.20 34.00 38.40 TABLE 7 Statistical analysis of the data in Tables 5 6.

Treatment Time Baseline 1 2 3 4 Control Baseline 0.6175 0.7093 0.088 0.5189 0.8629 vs Control Significance ns ns ns ns ns Treated Basline 0.0322 0.0012 0.0042 0.0273 0.0043 vs Treated Significance 0.05 <0.002 <0.005 <0.05 <0.005 Treated vs 0.6343 0.7772 0.0442 0.0018 0.0535 0.0515 Control Significance ns ns <0.05 <0.002 ns ns SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCTICA96/00282 28 Expression of HLA-DR on peripheral blood mononuclear cells.

The proportion of cells expressing HLA-DR, measured as a percentage of all CD45+ mononuclear cells, is shown in Tables 8 9 and Table 10 shows a summary of the statistical analysis of these data. As observed with the expression of HLA-DR was significantly enhanced on each of the five occasions investigated during and after a course of VasoCare T therapy. Within the treated group, all post-treatment values were significantly higher than the pre-treatment baseline. As for CD25, no significant changes were observed in the control group. Comparison of the treated with the placebo group showed that, at posttreatment times 2, 3, 4, and 5, the treated group showed significantly greater HLA-DR expression than the control group.

TABLE 8 The proportion of cells expressing HLA-DR (as a percentage of CD45+ cells) in peripheral blood from treated volunteers before and after a course of treatment.

SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCTICA96/rj282 29 Treated PreTreatment Post-i Post-2 Post-3 Post-4 Subject No. (Baseline) 1 39 37 48 36 40 47 2 15 37 33 35 54 82 3 49 61 52 52 96 49 4 8 32 31 39 14 17 26 57 76 92 30 62 6 41 50 63 61 54 54 7 46 53 73 56 54 62 8 39 37 48 36 40 47 9 41 44 45 70 55 28 21 52 37 28 11 34 34 45 54 50 49 12 48 44 71 35 53 13 25 42 35 75 54 91 14 45 48 52 59 61 Mean T 34.57 42.64 51.71 52.64 48.79 58.57 TABLE 9 The proportion of cells percentage of CD45+ cells) in expressing HLA-DR (as a peripheral blood from placebo-treated (control) volunteers before course of injections of untreated blood.

and af ter a Control Pre- Post-i Post-2 Post-3 Post-4 Post-S Subject No. Treatnent (Baseline) 1 20 15 19 17 14 24 2 39 44 42 32 31 41 3 28 36 30 33 33 47 4 33 30 37 39 47 56 55 33 43 51 35 Mean= 35.00 31.6 34.2 344 32.00 141.60 SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCrCA96/00292 30 TABLE Statistical analysis of the data in Tables 8 9.

Treatment Time Baseline 1 2 3 4 Control Baseline 0.5518 0.7978 0.8276 0.6331 0.3849 vs Control Significance NS NS NS NS NS Treated Basline 0.0223 0.0002 0.0072 0.0029 0.0008 vs Treated Significance <0.05 <0.0005 <0.01 <0.005 <0.001 Treated vs 0.9516 0.0871 0.0139 0.0309 0.0429 0.0355 Control Significance NS NS <0.02 <0.05 <0.05 <0.05 Expression of Ber-Mac 3 peripheral blood mononuclear cells.

The proportion of cells expressing Ber-Mac 3, a monocyte-specific surface marker, measured as a percentage of all CD45 positive mononuclear cells is shown in Tables 13 14 and Table 15 shows a summary of the statistical analysis of these data. As observed with the other leucocyte surface markers, the expression of Ber-Mac 3 was significantly enhanced during the course of therapy.

Within the treated group, post-therapy values were significantly higher than the pre-treatment baseline at times 1 2. As in the case of the other two markers investigated, no significant changes were observed in the control group. Comparisons of the treated with the placebo group showed only a single significant difference of Ber- Mac 3 expression, at time 2.

SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCTICA96/00282 31 TABLE 11 The proportion of cells expressing Ber-Mac 3 (as a percentage of CD45+ cells) in peripheral blood from treated volunteers before and after a course of therapy.

Treated PreTreatment Post-1 Post-2 Post-3 Post-4 Subject No. (Baseline) 1 41 37 52 25 34 47 2 19 31 37 32 34 18 3 39 39 44 46 61 47 4 39 42 55 60 33 19 40 47 34 37 16 6 46 49 52 35 38 7 30 30 35 34 37 46 8 38 36 45 34 45 44 9 38 42 37 40 32 30 28 47 38 27 34 11 34 58 35 44 44 12 37 48 47 32 56 13 29 38 58 69 32 47 14 33 29 28 35 35 26 Mean 33.71 39.07 44.21 39.86 38.93 4036 TABLE 12 The proportion of cells expressing Ber-Mac 3 (as a percentage of CD45+ cells) in peripheral blood from placebo-treated (control) volunteers before and after a course of injections of untreated blood.

SUBSTITUTE SHEET (RULE 26) WO 96/34613 PCT/CA96/00282 32 Control Pre- Post-i Post-2 Post-3 Post-4 Post-S Subject No. Treatment (Baseline)_ 1 29 21 25 27 26 31 2 30 27 36 41 49 43 3 35 76 38 33 54 68 4 19 21 36 29 39 64 38 20 38 36 23 22 Mean =30.20 33.00 34.60 33.20 38.20 45.60 TABLE 13 Statistical analysis of the data in Tables 11 and 12.

Treatment Time Baseline 1 2 3 4 Control Baseline 0.7955 0.2839 0.3832 0.3286 0.2290 vs Control Significance as as as ns 115 Treatment 0.0427 0.0019 0.1255 0.0845 0.0915 Baseline vs TreatedI Significance p <0.002 as ns ns Treated vs 0.3889 0.6122 0.0147 0.1186 0.9167 0.6160 Control Significance as as p <0.0 2 as as a s SUBSTITUTE SHEET (RULE 26)

Claims (49)

1. An aliquot of blood from a mammalian patient, said aliquot having been subjected to at least one stressor selected from elevated temperature in the range from 37 0 C to 0 C, ultraviolet radiation and oxidative environment, characterized by the ability, after injection into the patient's bloodstream, to create in the patient's peripheral blood a population of stimulated leukocytes having upregulated expression of CD25 cell surface markers, and the ability to enhance endothelial function in the patient.

2. An aliquot of mammalian blood as claimed in claim 1, further characterized by ability to create in the patient's peripheral blood after injection thereto a population of stimulated leukocytes having upregulated expression of HLA-DR cell surface markers.

3. An aliquot of mammalian blood as claimed in claim 1 or claim 2 further characterized by ability to create in the patient's peripheral blood after injection thereto a population of stimulated leukocytes capable of increasing the production in the patient's blood vessels of prostacyclin.

4. An aliquot of mammalian blood as claimed in any preceding claim which has been subjected to each said stressor simultaneously. An aliquot of mammalian blood as claimed in claim 4 which has been subjected to an oxidative environment stressor comprising the application thereto of gaseous oxidizing agent comprising a mixture of medical grade oxygen and ozone.

SUBSTITUTE SHEET (RULE 26) 34

6. An aliquot of mammalian blood as claimed in claim wherein the gaseous oxidizing mixture comprises from 1.0-100 Lg per mL, applied to the aliquot at a rate of from 0.01-2.0 litres/minute.

7. An aliquot of mammalian blood as claimed in claim 6 which has been subjected to an ultraviolet radiation stressor comprising output from a UV lamp emitting in the C-band wavelengths.

8. An aliquot of mammalian blood according to any preceding claim which is human blood, and which has a volume of from 0.1-100 mLs.

9. An aliquot of mammalian blood according to any preceding claim which has been subjected to said stressors for a period of time of from 0.5-60 minutes.

10. Use in preparation of a medicament for treatment of a vasospastic disorder in a human patient, of an aliquot of the patient's blood which has been subjected extracorporeally to at least one stressor selected from elevated temperature in the range 37 0 -55 0 C, ultraviolet 20 radiation and oxidative environment.

11. Use according to claim 10, wherein the aliquot of blood has been subjected to all three of said stressors simultaneously. a

12. use according to claim 10 or claim 11, wherein the oxidative environment stressor is a gaseous oxidizing agent including a mixture of medical grade oxygen and ozone.

13. Use according to claim 12 wherein the gaseous oxidizing mixture includes an ozone content of 1.0-100 lg per mL, applied to the aliquot at a rate of from 0.01-2.0 litres/minute.

14. Use according to any one of claims 10 to 13, R wherein the ultraviolet radiation stressor includes output H:\WendyS\Keep\species\54944-96 Vasogen.doc 19/04/00 35 from a UV lamp emitting in the C-band wavelengths.

Use according to any one of claims 10 to 14, wherein the aliquot has a volume of from 0.01-100mLs.

16. Use according to any one of claims 10 to wherein the aliquot has been subjected to said stressor(s) for a period of time of from 0.5-60 minutes.

17. Use according to any one of claims 10 to 16, wherein the vasospastic disorder is Raynaud's.

18. Use according to any one of claims 10 to 16, wherein the vasospastic disorder is migraine.

19. Use according to any one of claims 10 to 16, wherein the vasospastic disorder is cluster headache.

Use according to any one of claims 10 to 16, wherein the vasospastic disorder is syndrome X. 15

21. Use according to claim 10, substantially as described herein with reference to the examples and drawings. i

22. A method of alleviating the symptoms of a vasospastic disorder in a human patient, which includes the 20 steps of: extracting an aliquot of blood from a patient suffering from the disorder; subjecting the aliquot of blood, extracorporeally, to at least one stressor selected from 25 elevated temperature of from 37 0 -55 0 C, ultraviolet radiation and oxidative environment; and re-injecting the blood aliquot into the patient.

23. A process according to claim 22, wherein the aliquot is subjected to each said stressor simultaneously. H:\WendyS\Keep\species\54944-96 Vasogendoc 19/04/00 36

24. A process according to claim 22 or claim 23, wherein the oxidative environment stressor is a gaseous oxidizing agent including a mixture of medical grade oxygen and ozone.

25. A process according to claim 24, wherein the gaseous oxidizing mixture includes an ozone content of from 1.0-100Lg/mL, applied to the aliquot at a rate of from 0.01-2.0 litres/minute.

26. A process according to any one of claims 22 to 25, wherein the ultraviolet radiation stressor includes output from a UV lamp emitting in the C-band wavelengths.

27. A process according to any one of claims 22 to 26, wherein the aliquot of blood has a volume of from 0.1- 100mLs.

28. A process according to any one of claims 22 to 27, wherein the aliquot is subjected to said stressor(s) for a period of time of from 0.5-60 minutes.

29. A process according to any one of claims 22 to 28, wherein the vasospastic disorder is Raynaud's disease. S 20

30. A process according to any one of claims 22 to 28, wherein the vasospastic disorder is migraine.

S31. A process according to any one of claims 22 to 28, wherein the vasospastic disorder is cluster headache.

32. A process according to any one of claims 22 to 25 28, wherein the vasospastic disorder is syndrome X.

33. A method according to claim 22 for alleviating the symptoms of a vasospastic disorder, substantially as described herein with reference to the examples and drawings.

34. Use in the preparation of a medicament for treatment of atherosclerosis in a mammalian subject, of an aliquot of the subject's blood which has been subjected H:\WendyS\Keep\species\54944-96 Vasogendoc 19/04/00 37 extracorporeally to at least one stressor selected from elevated temperature in the range 37 0 -55 0 C, ultraviolet radiation and oxidative environment.

Use according to claim 34, wherein the aliquot of blood has been subjected to all three of said stressors simultaneously.

36. Use according to claim 34 or claim 35, wherein the oxidative environment stressor is a gaseous oxidizing agent including a mixture of medical grade oxygen and ozone.

37. Use according to claim 36 wherein the gaseous oxidizing mixture includes an ozone content of 100~g/mL, applied to the aliquot at a rate of from 0.01-2.0 litres/minute.

38. Use according to any one of claims 34 to 37, wherein the ultraviolet radiation stressor includes output from a UV lamp emitting in the C-band wavelengths. a

39. Use according to any of claims 34 to 38, wherein the aliquot has a volume of from 0.01-100mLs. a S 20

40. Use according to any of claims 34 to 39, wherein the aliquot has been subjected to said stressor(s) for a period of time of from 0.5-60 minutes.

41. Use according to claim 34, substantially as described herein with reference to the examples and drawings.

42. A method of alleviating the symptoms of atherosclerosis in a mammalian subject, which includes: extracting an aliquot of blood from a subject suffering from atherosclerosis; subjecting the aliquot of blood, extracorporeally, to at least one stressor selected from elevated temperature of from 37 0 -55 0 C, ultraviolet H:\WendyS\Keep\species\ 5 4 9 4 4 -96 Vasogen.doc 19/04/00 38 radiation and oxidative environment; and re-injecting the blood aliquot into the subject.

43. A process according to claim 42, wherein the aliquot is subject to each said stressor simultaneously.

44. A process according to claim 42 or claim 43, wherein the oxidative environment stressor is a gaseous oxidizing agent including a mixture of medical grade oxygen and ozone.

45. A process according to claim 44, wherein the gaseous oxidizing mixture includes an ozone content of from 1.0-100 g/mL, applied to the aliquot at a rate of from 0.01-2.0 litres/minute.

46. A process according to any of claims 42 to 15 herein the ultraviolet radiation stressor includes output from a UV lamp emitting in the C-band wavelengths.

47. A process according to any of claims 42 to 46, wherein the aliquot of blood has a volume of from 0.1- 100mLs.

48. A process according to any of claims 42 to 47, wherein the aliquot is subjected to said stressor(s) for a period of time of from 0.5-60 minutes.

49. A method according to claim 42, substantially as described herein with reference to the example and 25 drawings. H:\WendyS\Keep\species\54944-96 Vasogen.doc 19/04/00 39 An aliquot of blood according to claim 1, substantially as described herein with reference to the examples and drawings. Dated this 19th day of April 2000 VASOGEN INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\WendyS\Keep\species\54944-96 vasogen.doc 19/04/00